Method and system for automated focus-distance determination for molecular array scanners

ABSTRACT

An automated method and system for determining an optimal focus distance for scanning a molecular array scanner. Blocks of rows of a reference array are automatically scanned at successively greater distances of the stage from a light gathering medium, such as an optical fiber, or z-positions, to produce data providing a functional relationship between z-position and measured signal intensities. The data is then processed by a peak-height-based, or window-based, focus-finding routine that selects an optimal focus-distance for data scans.

TECHNICAL FIELD

[0001] The present invention relates to the molecular array scannersand, in particular, to automated adjustment of the focal distance atwhich a sample molecular array is positioned with respect to laser lightsources and photodetectors to produce optimal data acquisition.

BACKGROUND OF THE INVENTION

[0002] The present invention is related to acquisition ofmolecular-array data and other types of genetic, biochemical, andchemical data from molecular arrays by molecular array scanners. Ageneral background of molecular-array technology is first provided, inthis section, to facilitate discussion of the scanning techniquesdescribed in following sections.

[0003] Array technologies have gained prominence in biological researchand are likely to become important and widely used diagnostic tools inthe healthcare industry. Currently, molecular-array techniques are mostoften used to determine the concentrations of particular nucleic-acidpolymers in complex sample solutions. Molecular-array-based analyticaltechniques are not, however, restricted to analysis of nucleic acidsolutions, but may be employed to analyze complex solutions of any typeof molecule that can be optically or radiometrically scanned and thatcan bind with high specificity to complementary molecules synthesizedwithin, or bound to, discrete features on the surface of an array.Because arrays are widely used for analysis of nucleic acid samples, thefollowing background information on arrays is introduced in the contextof analysis of nucleic acid solutions following a brief background ofnucleic acid chemistry.

[0004] Deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) arelinear polymers, each synthesized from four different types of subunitmolecules. The subunit molecules for DNA include: (1) deoxy-adenosine,abbreviated “A,” a purine nucleoside; (2) deoxy-thymidine, abbreviated“T,” a pyrimidine nucleoside; (3) deoxy-cytosine, abbreviated “C,” apyrimidine nucleoside; and (4) deoxy-guanosine, abbreviated “G,” apurine nucleoside. The subunit molecules for RNA include: (1) adenosine,abbreviated “A,” a purine nucleoside; (2) uracil, abbreviated “U,” apyrimidine nucleoside; (3) cytosine, abbreviated “C,” a pyrimidinenucleoside; and (4) guanosine, abbreviated “G,” a purine nucleoside.FIG. 1 illustrates a short DNA polymer 100, called an oligomer, composedof the following subunits: (1) deoxy-adenosine 102; (2) deoxy-thymidine104; (3) deoxy-cytosine 106; and (4) deoxy-guanosine 108. Whenphosphorylated, subunits of DNA and RNA molecules are called“nucleotides” and are linked together through phosphodiester bonds110-115 to form DNA and RNA polymers. A linear DNA molecule, such as theoligomer shown in FIG. 1, has a 5′ end 118 and a 3′ end 120. A DNApolymer can be chemically characterized by writing, in sequence from the5′ end to the 3′ end, the single letter abbreviations for the nucleotidesubunits that together compose the DNA polymer. For example, theoligomer 100 shown in FIG. 1 can be chemically represented as “ATCG.” ADNA nucleotide comprises a purine or pyrimidine base (e.g. adenine 122of the deoxy-adenylate nucleotide 102), a deoxy-ribose sugar (e.g.deoxy-ribose 124 of the deoxy-adenylate nucleotide 102), and a phosphategroup (e.g. phosphate 126) that links one nucleotide to anothernucleotide in the DNA polymer. In RNA polymers, the nucleotides containribose sugars rather than deoxy-ribose sugars. In ribose, a hydroxylgroup takes the place of the 2′ hydrogen 128 in a DNA nucleotide. RNApolymers contain uridine nucleosides rather than the deoxy-thymidinenucleosides contained in DNA. The pyrimidine base uracil lacks a methylgroup (130 in FIG. 1) contained in the pyrimidine base thymine ofdeoxy-thymidine.

[0005] The DNA polymers that contain the organization information forliving organisms occur in the nuclei of cells in pairs, formingdouble-stranded DNA helixes. One polymer of the pair is laid out in a 5′to 3′ direction, and the other polymer of the pair is laid out in a 3′to 5′ direction. The two DNA polymers in a double-stranded DNA helix aretherefore described as being anti-parallel. The two DNA polymers, orstrands, within a double-stranded DNA helix are bound to each otherthrough attractive forces including hydrophobic interactions betweenstacked purine and pyrimidine bases and hydrogen bonding between purineand pyrimidine bases, the attractive forces emphasized by conformationalconstraints of DNA polymers. Because of a number of chemical andtopographic constraints, double-stranded DNA helices are most stablewhen deoxy-adenylate subunits of one strand hydrogen bond todeoxy-thymidylate subunits of the other strand, and deoxy-guanylatesubunits of one strand hydrogen bond to corresponding deoxy-cytidilatesubunits of the other strand.

[0006] FIGS. 2A-B illustrate the hydrogen bonding between the purine andpyrimidine bases of two anti-parallel DNA strands. FIG. 2A showshydrogen bonding between adenine and thymine bases of correspondingadenosine and thymidine subunits, and FIG. 2B shows hydrogen bondingbetween guanine and cytosine bases of corresponding guanosine andcytosine subunits. Note that there are two hydrogen bonds 202 and 203 inthe adenine/thymine base pair, and three hydrogen bonds 204-206 in theguanosine/cytosine base pair, as a result of which GC base pairscontribute greater thermodynamic stability to DNA duplexes than AT basepairs. AT and GC base pairs, illustrated in FIGS. 2A-B, are known asWatson-Crick (“WC”) base pairs.

[0007] Two DNA strands linked together by hydrogen bonds forms thefamiliar helix structure of a double-stranded DNA helix. FIG. 3illustrates a short section of a DNA double helix 300 comprising a firststrand 302 and a second, anti-parallel strand 304. The ribbon-likestrands in FIG. 3 represent the deoxyribose and phosphate backbones ofthe two anti-parallel strands, with hydrogen-bonding purine andpyrimidine base pairs, such as base pair 306, interconnecting the twostrands. Deoxy-guanylate subunits of one strand are generally pairedwith deoxy-cytidilate subunits from the other strand, anddeoxy-thymidilate subunits in one strand are generally paired withdeoxy-adenylate subunits from the other strand. However, non-WC basepairings may occur within double-stranded DNA.

[0008] Double-stranded DNA may be denatured, or converted into singlestranded DNA, by changing the ionic strength of the solution containingthe double-stranded DNA or by raising the temperature of the solution.Single-stranded DNA polymers may be renatured, or converted back intoDNA duplexes, by reversing the denaturing conditions, for example bylowering the temperature of the solution containing complementarysingle-stranded DNA polymers. During renaturing or hybridization,complementary bases of anti-parallel DNA strands form WC base pairs in acooperative fashion, leading to reannealing of the DNA duplex. StrictlyA-T and G-C complementarity between anti-parallel polymers leads to thegreatest thermodynamic stability, but partial complementarity includingnon-WC base pairing may also occur to produce relatively stableassociations between partially-complementary polymers. In general, thelonger the regions of consecutive WC base pairing between two nucleicacid polymers, the greater the stability of hybridization between thetwo polymers under renaturing conditions.

[0009] The ability to denature and renature double-stranded DNA has ledto the development of many extremely powerful and discriminating assaytechnologies for identifying the presence of DNA and RNA polymers havingparticular base sequences or containing particular base subsequenceswithin complex mixtures of different nucleic acid polymers, otherbiopolymers, and inorganic and organic chemical compounds. One suchmethodology is the array-based hybridization assay. FIGS. 4-7 illustratethe principle of the array-based hybridization assay. An array (402 inFIG. 4) comprises a substrate upon which a regular pattern of featuresare prepared by various manufacturing processes. The array 402 in FIG.4, and in subsequent FIGS. 5-7, has a grid-like two-dimensional patternof square features, such as feature 404 shown in the upper left-handcomer of the array. It should be noted that many molecular arrayscontain disk-shaped features, rather than round features. Each featureof the array contains a large number of identical oligonucleotidescovalently bound to the surface of the feature. These boundoligonucleotides are known as probes. In general, chemically distinctprobes are bound to the different features of an array, so that eachfeature corresponds to a particular nucleotide sequence. In FIGS. 4-6,the principle of array-based hybridization assays is illustrated withrespect to the single feature 404 to which a number of identical probes405-409 are bound. In practice, each feature of the array contains ahigh density of such probes but, for the sake of clarity, only a subsetof these are shown in FIGS. 4-6.

[0010] Once an array has been prepared, the array may be exposed to asample solution of target DNA or RNA molecules (410-413 in FIG. 4)labeled with fluorophores, chemiluminescent compounds, or radioactiveatoms 415-418. Labeled target DNA or RNA hybridizes through base pairinginteractions to the complementary probe DNA, synthesized on the surfaceof the array. FIG. 5 shows a number of such target molecules 502-504hybridized to complementary probes 505-507, which are in turn bound tothe surface of the array 402. Targets, such as labeled DNA molecules 508and 509, that do not contains nucleotide sequences complementary to anyof the probes bound to array surface, do not hybridize to generatestable duplexes and, as a result, tend to remain in solution. The samplesolution is then rinsed from the surface of the array, washing away anyunbound labeled DNA molecules. Finally, the bound labeled DNA moleculesare detected via optical or radiometric scanning. FIG. 6 shows labeledtarget molecules emitting detectable fluorescence, radiation, or otherdetectable signal. Optical scanning involves exciting labels of boundlabeled DNA molecules with electromagnetic radiation of appropriatefrequency and detecting fluorescent emissions from the labels, ordetecting light emitted from chemiluminescent labels. When radioisotopelabels are employed, radiometric scanning can be used to detect thesignal emitted from the hybridized features. Additional types of signalsare also possible, including electrical signals generated by electricalproperties of bound target molecules, magnetic properties of boundtarget molecules, and other such physical properties of bound targetmolecules that can produce a detectable

[0011]

[0012] signal. Optical, radiometric, or other types of scanning producean analog or digital representation of the array as shown in FIG. 7,with features to which labeled target molecules are hybridized similarto 706 optically or digitally differentiated from those features towhich no labeled DNA molecules are bound. In other words, the analog ordigital representation of a scanned array displays positive signals forfeatures to which labeled DNA molecules are hybridized and displaysnegative features to which no, or an undetectably small number of,labeled DNA molecules are bound. Features displaying positive signals inthe analog or digital representation indicate the presence of DNAmolecules with complementary nucleotide sequences in the original samplesolution. Moreover, the signal intensity produced by a feature isgenerally related to the amount of labeled DNA bound to the feature, inturn related to the concentration, in the sample to which the array wasexposed, of labeled DNA complementary to the oligonucleotide within thefeature.

[0013] Array-based hybridization techniques allow extremely complexsolutions of DNA molecules to be analyzed in a single experiment. Anarray may contain from hundreds to tens of thousands of differentoligonucleotide probes, allowing for the detection of a subset ofcomplementary sequences from a complex pool of different target DNA orRNA polymers. In order to perform different sets of hybridizationanalyses, arrays containing different sets of bound oligonucleotides aremanufactured by any of a number of complex manufacturing techniques.These techniques generally involve synthesizing the oligonucleotideswithin corresponding features of the array through a series of complexiterative synthetic steps, or depositing oligonucleotides isolated frombiological material.

[0014] As pointed out above, array-based assays can involve other typesof biopolymers, synthetic polymers, and other types of chemicalentities. For example, one might attach protein antibodies to featuresof the array that would bind to soluble labeled antigens in a samplesolution. Many other types of chemical assays may be facilitated byarray technologies. For example, polysaccharides, glycoproteins,synthetic copolymers, including block copolymers, biopolymer-likepolymers with synthetic or derivitized monomers or monomer linkages, andmany other types of chemical or biochemical entities may serve as probeand target molecules for array-based analysis. A fundamental principleupon which arrays are based is that of specific recognition, by probemolecules affixed to the array, of target molecules, whether bysequence-mediated binding affinities, binding affinities based onconformational or topological properties of probe and target molecules,or binding affinities based on spatial distribution of electrical chargeon the surfaces of target and probe molecules.

[0015] An “array”, unless a contrary intention appears, includes anyone, two or three dimensional arrangement of addressable regions bearinga particular chemical moiety to moieties (for example, biopolymers suchas polynucleotide sequences) associated with that region. An array is“addressable” in that it has multiple regions of different moieties (forexample, different polynucleotide sequences) such that a region (a“feature” or “spot” of the array) at a particular predetermined location(an “address”) on the array will detect a particular target or class oftargets (although a feature may incidentally detect non-targets of thatfeature). Array features are typically, but need not be, separated byintervening spaces. In the case of an array, the “target” will bereferenced as a moiety in a mobile phase (typically fluid), to bedetected by probes (“target probes”) which are bound to the substrate atthe various regions. However, either of the “target” or “target probes”may be the one which is to be evaluated by the other (thus, either onecould be an unknown mixture of polynucleotides to be evaluated bybinding with the other). An “array layout” refers collectively to one ormore characteristics of the features, such as feature positioning, oneor more feature dimensions, and the chemical moiety or mixture ofmoieties at a given feature. “Hybridizing” and “binding”, with respectto polynucleotides, are used interchangeably.

[0016] Any given substrate may carry one, two, four or more or morearrays disposed on a front surface of the substrate. Depending upon theuse, any or all of the arrays may be the same or different from oneanother and each may contain multiple spots or features. A typical arraymay contain more than ten, more than one hundred, more than one thousandmore ten thousand features, or even more than one hundred thousandfeatures, in an area of less than 20 cm² or even less than 10 cm². Forexample, features may have widths (that is, diameter, for a round spot)in the range from a 10 μm to 1.0 cm. In other embodiments each featuremay have a width in the range of 1.0 μm to 1.0 mm, usually 5.0 μm to 500μm, and more usually 10 μm to 200 μm. Non-round features may have arearanges equivalent to that of circular features with the foregoing width(diameter) ranges. At least some, or all, of the features may be ofdifferent compositions (for example, when any repeats of each featurecomposition are excluded the remaining features may account for at least5%, 10%, or 20% of the total number of features). Interfeature areaswill typically (but not essentially) be present which do not carry anypolynucleotide (or other biopolymer of a type of which the features arecomposed). Such interfeature areas typically will be present where thearrays are formed by processes involving drop deposition of reagents butmay not be present when, for example, photolithographic arrayfabrication processes are used,. It will be appreciated though, that theinterfeature areas, when present, could be of various sizes andconfigurations.

[0017] The array features can have widths (that is, diameter, for around spot) in the range from a minimum of about 10 μm to a maximum ofabout 1.0 cm. In embodiments where very small spot sizes or featuresizes are desired, material can be deposited according to the inventionin small spots whose width is in the range about 1.0 μm to 1.0 mm,usually about 5.0 μm to 500 μm, and more usually about 10 μm to 200 μm.Features which are not round may have areas equivalent to the arearanges of round features 16 resulting from the foregoing diameterranges.

[0018] Each array may cover an area of less than 100 cm², or even lessthan 50, 10 or 1 cm². In many embodiments, the substrate carrying theone or more arrays will be shaped generally as a rectangular solid(although other shapes are possible), having a length of more than 4 mmand less than 1 m, usually more than 4 mm and less than 600 mm, moreusually less than 400 mm; a width of more than 4 mm and less than 1 m,usually less than 500 mm and more usually less than 400 mm; and athickness of more than 0.01 mm and less than 5.0 mm, usually more than0.1 mm and less than 2 mm and more usually more than 0.2 and less than 1mm. With arrays that are read by detecting fluorescence, the substratemay be of a material that emits low fluorescence upon illumination withthe excitation light. Additionally in this situation, the substrate maybe relatively transparent to reduce the absorption of the incidentilluminating laser light and subsequent heating if the focused laserbeam travels too slowly over a region. For example, substrate 10 maytransmit at least 20%, or 50% (or even at least 70%, 90%, or 95%), ofthe illuminating light incident on the front as may be measured acrossthe entire integrated spectrum of such illuminating light oralternatively at 532 nm or 633 nm.

[0019] Once the labeled target molecule has been hybridized to the probeon the surface, the array may be scanned by an appropriate technique,such as by optical scanning in cases where the labeling molecule is afluorophore or by radiometric scanning in cases where the signal isgenerated through a radioactive decay of labeled target. In the case ofoptical scanning, more than one fluorophore can be excited, with eachdifferent wavelength at which an array is scanned producing a differentsignal. In optical scanning, it is common to describe the signalsproduced by scanning in terms of the colors of the wavelengths of lightemployed for the scan. For example, a red signal is produced by scanningthe array with light having a wavelength corresponding to that ofvisible red light.

[0020] Scanning of a feature by an optical scanning device orradiometric scanning device generally produces a scanned imagecomprising a rectilinear grid of pixels, with each pixel having acorresponding signal intensity. These signal intensities are processedby an array-data-processing program that analyzes data scanned from anarray to produce experimental or diagnostic results which are stored ina computer-readable medium, transferred to an intercommunicating entityvia electronic signals, printed in a human-readable format, or otherwisemade available for further use. Molecular array experiments can indicateprecise gene-expression responses of organisms to drugs, other chemicaland biological substances, environmental factors, and other effects.Molecular array experiments can also be used to diagnose disease, forgene sequencing, and for analytical chemistry. Processing of moleculararray data can produce detailed chemical and biological analyses,disease diagnoses, and other information that can be stored in acomputer-readable medium, transferred to an intercommunicating entityvia electronic signals, printed in a human-readable format, or otherwisemade available for further use.

[0021]FIG. 8 illustrates components of a molecular array scanner. Lasers800 a-b emit coherent light that passes through electro-optic modulators(“EOMs”) 810 a-b with attached polarizers 820 a-b. Each EOM andcorresponding polarizer together act as a variable optical attenuator. Acontrol signal in the form of a variable voltage is applied to each EOM810 a-b by controller 880. The controller 880 may include a suitablyprogrammed processor, logic circuit, firmware, or a combination ofsoftware programs, logic circuits, and firmware. The control signalchanges the polarization of the laser light, which alters the intensityof the light that passes through the EOM. In general, laser 800 aprovides coherent light of a different wavelength than that provided bylaser 810 b. For example, one laser may provide red light and the otherlaser may provide green light. The beams may be combined along a pathtoward a stage 800 by the use of full mirror 851 and dichroic mirror853. The light from the lasers 800 a-b is then transmitted through adichroic beam splitter 854, reflected off fully reflecting mirror 856,and then focused, using optical components in beam focuser 860, onto amolecular array mounted on a holder 800. Fluorescent light, emitted attwo different wavelengths (for example, green light and red light) fromfeatures of the molecular array in response to illumination by the laserlight, is imaged using the optics in the focuser/scanner 860, and isreflected off mirrors 856 and 854. The two different wavelengths arefurther separated by a dichroic mirror 858 and are passed tophotodetectors 850 a-b. More optical components (not shown in FIG. 8)may be used between the dichroic mirror and the photodetectors 850 a-b,such as lenses, pinholes, filters, and fibers. The photodetectors 850a-b may be of various different types, including photo-multiplier tubes,charge-coupled devices, and avalanche photodiodes.

[0022] A scan system causes a light spot from each laser 800 a-b to bemoved in a regular pattern about the surface of the molecular array. Themolecular array is mounted to a stage that can be moved in horizontaland vertical directions to position light from the lasers onto aparticular region at the surface of the molecular array, from whichregion fluorescent emission is passed back to the photodetectors via theoptical path described above. An autofocus detector 870 is provided tosense and correct any offset between different regions of the moleculararray and the focal plane of the system during scanning. An autofocussystem includes detector 870, processor 880, and a motorized adjuster tomove the stage in the direction of arrow 896.

[0023] The controller 880 receives signals from photodetectors 850 a-b,called “channels,” corresponding to the intensity of the green and redfluorescent light emitted by probe labels excited by the laser light.The controller 880 also receives a signal from autofocus offset detector870 in order to control stage adjustment, provides the control signal tothe EOMs 810 a-b, and controls the scan system. Controller 880 may alsoanalyze, store, and output data relating to emitted signals receivedfrom detectors 850 a-b.

[0024] The dynamic autofocus mechanism, described above, provides ameans for maintaining a constant focus distance during scanning of amolecular array. However, any of various different focus distances maybe selected to be held constant during a scan. In general, the focusdistance must correspond to the depth of focus of the molecular arrayoptics, but, in general, there is one or a small range of optimal focusdistances for a particular molecular array scanner. Designers,manufacturers, and users of molecular arrays have recognized a need foran automated focus-distance optimization method.

SUMMARY OF THE INVENTION

[0025] One embodiment of the present invention provides an automatedmethod for determining an optimal focus distance for scanning amolecular array by a molecular array scanner. Blocks of rows of areference array are automatically scanned over a range of focusdistances, or z-positions, to produce data providing a functionalrelationship between focus distance, or z-position, and measured signalintensities. The data is then processed by a peak-height-based, or localvariation-based, focus-finding routine that selects an optimal focusdistance for data scans.

[0026] The present invention further provides a computer program productfor use with an apparatus such as described herein. The program productincludes a computer readable storage medium having a computer programstored thereon and which, when loaded into a programmable processor,provides instructions to the processor of that apparatus such that itwill execute the procedures required of it to perform a method of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 illustrates a short DNA polymer 100, called an oligomer,composed of the following subunits: (1) deoxy-adenosine 102; (2)deoxy-thymidine 104; (3) deoxy-cytosine 106; and (4) deoxy-guanosine108.

[0028] FIGS. 2A-B illustrate the hydrogen bonding between the purine andpyrimidine bases of two anti-parallel DNA strands.

[0029]FIG. 3 illustrates a short section of a DNA double helix 300comprising a first strand 302 and a second, anti-parallel strand 304.

[0030] FIGS. 4-7 illustrate the principle of the array-basedhybridization assay.

[0031]FIG. 8 is a block diagram of major optical and electroniccomponents of a molecular array scanner.

[0032]FIG. 9 abstractly illustrates the scanner components related tothe automated focus-distance-determination method that represents oneembodiment of the present invention.

[0033]FIG. 10 illustrates the effect of small changes in the z-positionof an illuminated spot on the surface of a molecular array with respectto the image of the emitted light from the illuminated spot of thesurface of the molecular array focused on the surface of an opticalfiber.

[0034]FIG. 11 shows a representation of the function of the signalintensity I measured by the photodetector of a molecular array scanneras a function of the z-position of an illuminated region of themolecular array that generates the signal.

[0035]FIG. 12 illustrates the variation in signal intensity I withz-position z in the vicinity of the peak of the I_(z) curve.

[0036]FIG. 13 illustrates selection of an optimal focus distance Z_(p).

[0037]FIG. 14 illustrates the signal intensity variation over az-position variation centered about the peak of the I/z curve.

[0038]FIG. 15 illustrates the scan pattern employed in a preferredembodiment of the present invention.

[0039]FIG. 16 is a flow-control diagram for the optimal focus-distancedetermination method “focus_distance” that represents one embodiment ofthe present invention.

[0040] FIGS. 17-21 illustrate operation of the routine“peak_intensity_focus” with reference to I/z functional curves.

[0041]FIG. 22 is a flow-control diagram for the routine“peak_intensity_focus.”

[0042] FIGS. 23-29 illustrate operation of window_focus using graphicalrepresentations of I/z curves.

[0043] FIGS. 30-31 show flow-control diagrams that describe operation ofwindow_focus.

DETAILED DESCRIPTION OF THE INVENTION

[0044] One embodiment of the present invention is a method fordetermining an optimal focus distance at which to scan the surface of amolecular array during data collection from the molecular array by anautomated molecular array scanner. The molecular array scanner includesdynamic autofocus functionality for maintaining the focus distance at aconstant value during scanning of a molecular array. However, currentscanners lack an automated focus-distance optimizing feature, andinstead require a tedious and error-prone manual optimal focus-distancedetermination. The method for automated focus-distance determinationthat represents one embodiment of the present invention may beincorporated into logic circuits, firmware, or software, or acombination of logic circuits, firmware, and software, within thecontrol logic of a molecular scanner.

[0045]FIG. 9 abstractly illustrates the scanner components related tothe automated focus-distance determination method that represents oneembodiment of the present invention. A molecular array 902 is mounted tothe stage 904 of a molecular array scanner. The stage 904 can betranslated vertically and horizontally in a plane coincident with theplane of the surface of the stage by X and Y translation mechanisms notshown in FIG. 9. In addition, the distance of the stage 904 from theoptical fiber or other light-gathering medium can be controlled by adistance-control mechanism that moves the stage and stage support 906 ina direction perpendicular to the plane of the scanner.

[0046] Light from a laser 908, focused and filtered via various opticcomponents 910, illuminates a small region of the array 912 from whichlight of a generally longer wavelength is emitted by fluorescent orchemiluminescent compounds incorporated into target molecules. Theemitted light is filtered and focused by various optical components 914and 916 into a roughly disk-shaped spot 918 that impinges on the surfaceof an optical fiber 920 or another light-collection medium that inputsemitted light from the surface of the molecular array into aphotodetector 922.

[0047] An orthogonal x, y, z coordinate system 924 is shown near theleft, lower corner of the molecular array-scanner stage 904 to indicatethe directions in which the stage can be moved relative to the surfaceof the optical fiber 920 on which emitted light is focused. The exactfunctional relationship between variations in the distance from theemitted-light source to the surface of the optical fiber 920, closelyrelated to the distance of the stage from the optical fiber 920,referred to as the z-position position of the stage, depends on thegeometry of the laser, molecular array, and optical fiber geometries.However, the focus-distance is a continuous function of z-position. Ifthe plane of the surface of the molecular array 902 is not parallel tothe plane of the surface of the stage 904, then horizontal translationsin the x, y plane of the stage may also affect the distance betweenlight-emitting probe molecules on the surface of the molecular array andthe optical fiber 920. The dynamic autofocus feedback-control mechanismwithin this scanner can dynamically adjust the z-position of the stageas the stage is translated in the x, y plane in order to correct for thesmall focus-distance variation during a scan due tomolecular-array-surface irregularities and positioning and orientationproblems that cause the plane of the surface of the molecular array tobe inclined with respect to the plane of the surface of the stage. Thus,when an optimal focus-distance is determined, a molecular array can bescanned at this focus distance by the autofocus mechanisms within thescanner. Because the focus distance is a continuous function of thez-coordinate of the spatial location of the illuminated spot 912 of themolecular-array surface, which is, in turn, a continuous function of thez-position of the stage, the following discussion is related tooptimizing the z-position of the stage 904. It should be noted that thefollowing discussion and Figures could also be cast in terms ofz-coordinate of the laser- illuminated region of a molecular array, orthe distance of either the stage or the laser-illuminated region of amolecular array from the emitted-light-collection medium.

[0048]FIG. 10 illustrates the effect of small changes in the z-positionof the stage, or z-coordinate of an illuminated spot on the surface of amolecular array, with respect to the position of the image of theemitted light from the illuminated spot of the surface of the moleculararray focused onto the surface of an optical fiber. This is one exampleof how the measured intensity of the emitted light can vary with changesin the focal distance. Another example is the loss of emission-intensitydue to the lack of correct focus of the illuminated spot on the surfaceof the molecular array.

[0049] In FIG. 10, the circular cross section of the surface of anoptical fiber (920 in FIG. 9) is represented by circles 1002-1007. Theimage of an illuminated spot on the surface of the molecular array isshown in FIG. 10 by various differently sized, cross-hatch-filledcircles 1008-1013. In FIG. 10, representations 1014-1019 of the focusingof the emitted light onto the plane of the circular cross section of thesurface of the optical fiber are shown at the left. In eachrepresentation, a lens-like optic, e.g. lens-like optic 1020, focuseslight to a point, e.g. point 1021, above, on, or below the plane, e.g.plane 1022, of the surface of the optical fiber. As the illuminated spoton the surface of the molecular array is moved in the z direction, theimage of the illuminated spot focused onto the surface of the opticalfiber, first defocused as illuminated spot 1008, focuses and decreasesin diameter relative to the diameter of the cross section of the opticalfiber. Illuminated spot 1010 represents a focused spot, corresponding tothe optics representation 1016. As the illuminated spot on the surfaceof the molecular array is moved further in the z direction, theilluminated spot gradually defocuses and grows in diameter relative tothe diameter of the cross section of the optical fiber. At eitherextreme of the z-position range, as indicated by optics representations1014 and 1019, the image of the illuminated spot is sufficientlydefocused that the image of the illuminated spot is larger than thecross section of the optical fiber, and emission photons representing aportion of the emitted-light are lost, resulting in a decrease in theintensity of the resulting measured signal. In FIG. 10, the z-positionsthat result in the corresponding respective sizes of the image of theilluminated spot and the cross section of the optical fiber plottedalong a coordinate axis 1024 corresponding to the z-position of thestage of the molecular scanner. The z-positions range from a lowestvalue, Z_(max) ⁻, to a highest z value, Z_(max) ⁺. In FIG. 10, forexample, the z-position of the stage resulting in a centered, focusedimage of the illuminated spot on the surface of the optical fiber 1026is shown with z coordinate 1028. In FIG. 10, a vertical coordinate axis1030 is shown orthogonal to the z-position coordinate axis 1024. Thisorthogonal coordinate axis corresponds to the intensity of the emittedlight detected by a photodetector (922 in FIG. 9) of the molecular arrayscanner. In FIG. 10, the horizontal lines, such as horizontal line 1032,plotted along the vertical z-position axis represent the signalintensities measured for a particular region of the surface of themolecular array corresponding to the z-positions of the stage of themolecular array. Again, as the image of the illuminated region of themolecular array defocuses past the edges of the fiber, not all of theemitted light is captured by the fiber and, as a result, the intensities1034-1036 of the signal level decreases proportionally to the fractionof light that is missing the fiber.

[0050] The graph of signal intensity to z-position, as shown in FIG. 10,can be rotated 90 degrees and plotted as a continuous, rather than adiscrete, function. FIG. 11 shows a representation of the function ofthe signal intensity I measured by the photodetector of a moleculararray scanner as a function of the z-position of an illuminated regionof the molecular array that generates the signal. The functionillustrated in FIG. 11 1102 is exaggerated in order to clearlyillustrate the general features of a typicalsignal-intensity-to-z-position (“I/z”) relationship for one class ofmolecular array scanners. As in FIG. 10, the functional relationship isplotted from a Z_(max) ⁻ position to a Z_(max) ⁺ position along thez-position axis 1104, with the height of the curve representing themeasured signal intensity with respect to the signal-intensity axis1106. Proceeding rightward from the Z_(max) ⁻ position 1108 towards tothe Z_(max) ⁻ position 1110, the signal intensity sharply rises to asignal intensity peak 1112 and then falls gradually to the edge 1114 ofa steeply descending curve that falls back to nearly 0 intensity atZ_(max) ³⁰ . In one class of molecular array scanners, the I/z functionappears as a mesa, with the top of the mesa (section of the curvebetween 1112 and 1114 in FIG. 11) slanted slightly downward.

[0051] The autofocus mechanism within a molecular array scanner, as withall electromechanical devices, is not infinitely precise. Instead, theautofocus mechanism is capable of maintaining the distance of theilluminated portion of a molecular array surface within a small range ofdistances by controlling the z-position of the stage. Thus, despite theautofocus mechanism, the z-position, functionally related to the focusdistance, varies slightly over the course of a molecular array scan.FIG. 12 illustrates the variation in signal intensity I with z-positionz in the vicinity of the peak of the I curve. In FIG. 12, the zcoordinate for the peak, z_(p), is plotted on the z axis 1202. The totalvariation in z-position during a scan is shown by the interval Δz 1204plotted from z_(l) to z_(r) on the z axis 1206-1207. The signalintensity varies over the interval Δz 1204 by a corresponding signalintensity interval ΔI. Note that, for the typical I/z functionalrelationship illustrated in FIG. 12, the signal intensity varies overthe entire ΔI interval 1208 within the portion of the Δz interval to theleft of the peak position Z_(p), while varying only relatively slightlyin the portion of the Δz interval to the right of the peak positionZ_(p). Thus, because of the asymmetry of the I/z curve with respect tothe peak position Z_(p), small fluctuations in the focus distancecorresponding to Z_(p) may result in relatively large variation insignal intensity.

[0052] It is desirable to select a focus distance that provides arelatively slight variation in signal intensity corresponding to slightvariations in focus distance during scanning of a molecular array. It isthe goal of an autofocus mechanism to help maintain a constant systemsensitivity for a constant quantity of flurophore over the entiresurface of a molecular array. FIG. 13 illustrates selection of anoptimal focus distance Z_(f). As shown in FIG. 13, if the optimal focusdistance Z_(f) is chosen roughly centered in the interval representingthe top of the mesa of the I/z curve, producing a Δz interval 1302within the top of the mesa, then the variation in signal intensity ΔI1304 over the interval Δz is relatively small compared with the ΔIinterval (1208 in FIG. 12) resulting from selection of the focusdistance at the peak of the I/z curve Z_(p).

[0053] If, by contrast, the scanner geometry and photo-detectionapparatus produces a symmetrical, Gaussian-like I/z functional curve,selection of a focus distance corresponding to the z-position of thecurve peak Z_(p) may result in the smallest possible signal intensityvariation. FIG. 14 illustrates the signal intensity variation over az-position variation centered about the peak of the I/z curve.

[0054] Thus, either a focus distance corresponding to a z-position at apositive offset from the peak z-position may be selected, for I/z curveshaving slanted mesa forms, or a focus distance corresponding to the peakz-position may be optimal for symmetrical, Gaussian-like I/z functionalrelationships. In either case, the optimal focus distance is a focusdistance that gives a relatively large signal intensity that variesrelatively slightly with variation in the focus distance.

[0055] The method for optimal focus distance selection that representsone embodiment of the present invention relies on a single-pass,automated scan of a reference molecular array. The reference moleculararray is prepared by precisely coating the surface of a molecular arraysubstrate with a polymethylmethacrylate polymer (“PMMA”) containing afluorescent or chemiluminescent dye. The PMMA polymer can be spun ontothe surface of a slide and, if necessary, planarized, usingPMMA-substrate-preparation techniques commonly employed in themanufacture of semiconductors. PMMA-based reference arrays are far moreuniform than previously employed cyanine-dye reference arrays. The U.S.patent application Ser. No. 10/008598 entitled “Devices For CalibratingOptical Scanners And Methods Of Using The Same” by Holcomb et al. (filedDec. 4, 2001) details the PMMA-based reference arrays, and is hereinincorporated by reference.

[0056]FIG. 15 illustrates the scan pattern employed in a preferredembodiment of the present invention. In FIG. 15, a molecular array 1502is abstractly represented with horizontal rows, such as row 1504. Thescan pattern involves scanning a fixed number of contiguous rows n ateach z-position over a range of z-positions Z_(max) ⁻ to Z_(max) ⁺. Thez-position is incremented by an increment i over this range ofz-positions. For example, as shown in FIG. 15, a contiguous block of nrows is first scanned at z-position z=−7i, a second block of ncontiguous rows is scanned at z-position z=−6I 1508, and so on, upthrough scanning of the continuous block of n rows 1510 at z-positionz=7i. The number of rows scanned at each z-position n, the z-positionincrement i, and the range Z_(max) ⁻ to Z_(max) ⁺ may all be specifiedin a configuration file for the automated focus-distance determination.Note that there are many alternative possiblefocus-distance-determination scan patterns. In one embodiment of thepresent invention, z positions ranging from −20μ (microns) to +20μ arescanned at 0.25μ increments, with ten rows scanned for each z-position.

[0057] In the following discussion, the described embodiment relates toa two-channel molecular array scanner that measures emitted light in redand green regions of the visible spectrum. Thus, the followingdiscussion refers to red and green channels and red and green signals.However, the present invention is equally applicable to single-channelmolecular array scanners or to molecular array scanners that measuremore than two types of signals, or measure signals from different partsof the electromagnetic radiation spectrum.

[0058]FIG. 16 is a flow-control diagram for the optimal focus-distancedetermination method “focus_distance” that represents one embodiment ofthe present invention. In step 1602, the scanner is initialized.Initialization involves checking the scanner to make sure that thescanner is properly physically configured, setting the photodetectorvoltages to default voltages, initializing a stage motion controller(s),and loading the reference molecular array into the scanner andverifying, via a bar code imprinted on the surface of the moleculararray, that the reference molecular array is the appropriate referencemolecular array for the optimal focus-distance determination. In step1604, the optimal focus-distance determination configuration file isinput, specifying various parameters for the optimal focus-distancedetermination, including the parameters n, i, Z_(max) ⁻ and Z_(max) ⁻parameters described with reference to FIG. 15, above. In step 1606, thereference array is scanned as specified by the configuration-fileparameters input in step 1604. The reference-array scan results in acomputer encoding of signal intensities measured for discrete regionscovering the surface of the molecular array. In many molecular arrayscanners, these signal-intensity results are encoded as integer orfloating-point values associated with pixels within the scanned image ofthe molecular array. In the for-loop comprising steps 1608, 1610, and1612, focus_distance filters and averages the signal intensities foreach block of n rows. The rows are filtered to remove saturated pixels,or, in other words, pixels with intensity values that exceed the linearrange of the electronics (due, for example, to dust), and the signalintensities of the remaining pixels are averaged to produce an averageintensity value corresponding to the z-position at which the rows werescanned. Note that, if the number of saturated pixels exceeds somespecified threshold, the scan may be rejected, and an indicationdisplayed by the molecular array scanner to a user indicating that themolecular array scanner needs to be reconfigured in order to producesignal intensities within appropriate ranges.

[0059] Next, in step 1614, the average intensity values for eachz-position are assembled into a computer-encoded format corresponding tothe I/z graphs shown in FIGS. 11-13 and 14, above. Note that each typeof signal, such as the red and green signals described above, in thebackground section, are measured to produce separate I/z functionalrelationship for each type of signal, or channel. The computer-encodeddata representing the I/z functional relationship for each channel canthen be scanned with respect to z-position to determine the I and zcoordinates of the peak of the I/z curve. In step 1616, focus_distancedetermines whether the z-positions of the I/z peaks for all channelsfall within an acceptable range of z-position values. If not, thenfocus_distance displays an error message in step 1618 indicating theneed for resetting the coarse adjustment of the z-component of theautofocus mechanism, or another resetting or reconfiguration of themolecular array scanner, and then returns an error. If all the peaksfall within an acceptable z-position range, then a focus routine iscalled in step 1620. The focus routine called depends on the geometryand configuration of the molecular array scanner. For molecular arrayscanners that produce mesa-shaped I/z functional curves, the routine“window_focus” is called. Alternatively, if the molecular array scannerproduces symmetrical, Gaussian-like curves, as shown in FIG. 14, thenthe focus routine “peak_intensity_focus” is called. Molecular scannersthat produce other types of I/z curves may need other, specific focusroutines. The focus function called in step 1620 produces an optimalz-position corresponding to an optimal focus distance, Z_(f), that canbe used by the molecular array scanner for one or more data-acquisitionscans.

[0060] Next, in step 1622, focus_distance graphically displays theresults of the optimal focus-distance determination to a user. If theuser wishes they can review the displayed information and can inputacceptance or rejection of the optimal focus-distance determination. Ifthe method is found to be robust enough for any particular configurationof scanner, this step can be skipped and the process be fully automated.If the optimal focus-distance determination is accepted, as determinedin step 1624, then the optimal focus distance is stored intonon-volatile memory within the array scanner in step 1626, andfocus_distance returns success. The optimal focus distance may be storedin flash memory, for example, or in other non-volatile memory storage,such as a mass storage device. If the optimal focus distance isrejected, as determined in step 1624, then the user is prompted forinput as to whether to retry the optimal focus-distance determination.If the user elects not to retry the optimal focus-distance determinationthen focus_distance returns an error. Otherwise, control flows back tothe initial step, step 1602.

[0061] For molecular scanners that produce Gaussian-like, roughlysymmetrical I/z curves, as shown in FIG. 14, the focusing routine“peak_intensity_focus” is called from step 1620 of focus_distance,described with reference to FIG. 16. FIGS. 17-21 illustrate operation ofthe routine “peak_intensity_focus” with reference to I/z functionalcurves. FIG. 22 is a flow-control diagram for the routine“peak_intensity_focus.” Note that the I/z curves shown in FIGS. 17-21are mesa-shaped curves characteristic of one class of molecular arrayscanners. Although the routine “peak_intensity_focus” provides bestresults for symmetrical, Gaussian-like I/z curves, it can also beapplied to the mesa-shaped curves shown in FIGS. 17-21.

[0062]FIG. 17 is a graph of the I/z curve for the red channel 1702 andthe I/z curve for the green channel 1704 of a two-channel moleculararray scanner. The routine “peak_intensity_focus” first determines thez-position Z_(p) 1706 of the peak in intensity for the red-channel I/zcurve 1702, as shown in FIG. 17. Next, as shown in FIG. 18,peak_intensity_focus begins from the z-position Z_(max) ⁻ and proceedsrightward, as shown by arrow 1802, searching for a Z_(l) z-position atwhich the corresponding signal intensity rises to a threshold percentageof the signal intensity corresponding to the Z_(p) peak z-position.After finding the z-position Z_(l), peak_intensity_focus starts atz-position Z_(p) 1804 and proceeds rightward, as indicated by arrow1806, evaluating the signal intensities of successive z-positions untilpeak_intensity_focus identifies a position Z_(r) 1808 at which thecorresponding signal intensity falls below the threshold value. As shownin FIG. 19, the z-positions Z_(l) 1803 and Z_(r) 1808 define endpointsof a red-channel plateau interval P_(r) 1902 for the red-channel I/zcurve. In similar fashion, peak_intensity_focus applies theplateau-finding technique to identify a green-channel plateau intervalP_(g) shown as interval 1202 in FIG. 20. Finally, as shown in FIG. 21,peak_intensity_focus determines whether the overlap 2102 between thered-channel plateau interval P_(r) 1902 and green-channel plateauinterval P_(g) 2002 is greater than or equal to a threshold length, inmicrons, and determines whether or not each of plateau intervals P_(r)and P_(g) are also greater than or equal to a threshold width, inmicrons. In one embodiment, the threshold width for both the overlap2102 and the plateau intervals 1902 and 2002 is four microns. If theoverlap and plateau intervals do not meet the threshold requirements,then peak_intensity_focus returns an error value. Otherwise, in oneembodiment, peak_intensity_focus returns a focus Z_(f) corresponding tothe z-position of the midpoint of the red-channel plateau interval 1902.

[0063] The routine “peak_intensity_focus” is alternatively described bythe flow-control diagram of FIG. 22. In step 2202, peak_intensity_focusreceives the I/z for each channel scanned by the molecular arrayscanner. In the for-loop comprising steps 2204-2209,peak_intensity_focus determines the plateau interval for each channel.In step 2205, peak_intensity_focus determines the z-position Z_(p)corresponding to the peak intensity for the currently considered channelc. In steps 2206 and 2207, peak_intensity_focus determines the Z_(l) andZ_(r) z-positions for the plateau interval for the currently consideredchannel c, as described above with reference to FIGS. 18-19. Then, instep 2208, peak_intensity_focus reports and stores the plateau intervalP_(c) for the currently considered channel c. In step 2209,peak_intensity_focus determines whether or not the plateau for anotherchannel needs to be computed and, if so, control flows back to step2205. When all the plateau interval shave been computed,peak_intensity_focus, in step 2210, determines the overlap between thecalculated and stored plateau intervals for all channels considered inthe for-loop comprising steps 2204-2209. In step 2212,peak_intensity_focus determines whether or not each plateau interval isgreater than or equal to a threshold value, in one embodiment, 4microns. If not, then an error is returned. Otherwise, in step 2214,peak_intensity_focus determines whether or not the overlap, calculatedin step 2210, is greater than or equal to a threshold value, in oneembodiment, also 4 microns. If not, then an error is returned.Otherwise, a focus z-position Z_(f) is selected in step 2216. In oneembodiment, the midpoint of the red-channel plateau interval is selectedas the focus Z_(f). In other embodiments, a centroid z-position for theplateau intervals or for the overlap may be selected as the focus Z_(f).In all embodiments, peak_intensity_focus returns the final selectedfocus Z_(f) in step 2218.

[0064] An alternate focus-finding routine is the focus-finding routine“window_focus,” also called in step 1620 of the routine focus_distanceillustrated in FIG. 16. The routine “window_focus” is more suitable forthe mesa-shaped I/z curves characteristic of one class of moleculararray scanners, for reasons described above with respect to FIGS. 12 and13. FIGS. 23-29 illustrate operation of window_focus using graphicalrepresentations of I/z curves. FIGS. 30-31 show flow-control diagramsthat describe operation of window_focus.

[0065] In a first step, as shown in FIG. 23, window_focus computes thez-positions Z_(P) _(r) and Z_(P) _(g) 2302 and 2304 for the red-channelI/z curve 1306 and the green-channel I/z curve 1308, respectively. FIGS.23-29 assume a two-channel molecular array scanner. Then, window_focuschecks to make sure that these peak positions Z_(P) _(r) and Z_(P) _(g)fall within a range of z-position values 1310 and 1312 that represents80 percent of the range Z_(max) ⁺−Z_(max) ⁻. If the peak z-positionsZ_(P) _(r) and Z_(P) _(g) fall outside this range, an error is returned.

[0066] Next, window_focus divides the z-position range Z_(max) ⁻−Z_(max)⁻ into a number of evenly sized increments. In one embodiment of thepresent invention, each increment represents a ¼ micron change in thez-position. The window_focus algorithm places the left edge of a window,or z-position interval, at each discrete z-position increment, with thelast few increments near Z_(max) ⁺ omitted, as truncated windows wouldresult. Therefore, there nearly as many windows as there are depthsettings. FIG. 24 illustrates the windows initially constructed bywindow-focus. A first window is defined by vertical lines 2402-2403. Asecond window is defined by vertical dashed lines 2404-2405. A thirdwindow is defined by vertical dashed-and-crossed lines 2406-2407.

[0067] Initially, window_focus employs wider windows to determineapproximate plateau intervals for each channel. In one embodiment, aninitial 8 micron window is used. If the window-based plateau-intervaldetermination fails to work for wider windows, it is tried again fornarrower windows. FIG. 25 illustrates narrower windows. Theplateau-interval determination is illustrated in FIGS. 26 and 27 for redand green channels, respectively. In FIG. 26, window_focus starts fromthe Z_(max) ⁻ z-position and proceeds rightward, as indicated by arrow2602, determining a slope for each window. This slope can be calculatedas the difference between the largest and smallest signal intensity seeninside the window expressed as a percentage of maximum intensity for theentire I/z curve between Z_(max) ⁻ and Z_(max) ⁺. Various methods can beused to determine the slope for a window, including applying adifferential smoothing operator and then dividing the difference betweenthe highest and lowest intensity values within the window by the lengthof the window increment. Alternative techniques may also be employed.Window_focus determines the slope of all initial windows. Window_focuslooks for positions for which the difference between the maximum andminimum intensity values within the window are less than 2% of themaximum intensity found anywhere on the entire I/z curve. Ifwindow_focus finds at least one such window, it will return thez-position of the center of the window with the smallest intensityvariation. If window_focus does not find a window with less than a 2%difference, then window_focus tries again with smaller-sized windows, inone embodiment, 6-micron windows If window_focus fails to find a windowwith less than a 2% difference, then window_focus tries again with 4micron windows. If window_focus fails to find a 4-micron window withless than a 2% difference, then window_focus returns the 4-micron withthe smallest intensity variations, using an RMS-like intensity variationmeasure discussed below. In FIG. 26, window 2704 is shown as meeting thecriteria for a plateau window, although, as pointed out above, the slopeof the plateau in the figures is exaggerated, and thus appears to createan intensity variation greater than 2%.

[0068]FIG. 27 illustrates determination of the plateau window 2702 forthe green channel. Thus, by the window-based method, a red-channelplateau interval 2704 and a green-channel plateau interval 2702 aredetermined with respect to the z-position coordinate axis. As shown inFIGS. 28A-B, window_focus determines an overlap region common to thedetermined plateau intervals for each channel. In FIGS. 28A-B, theplateau windows for the red channel (2802 and 2804) are plottedhorizontally in z-position (with respect to the plateau windows for thegreen channel (2806 and 2808). Z axis segments (2810-2813) are plottedabove and below the plateau-window plots. In FIG. 28A, the overlapregion begins at z-position 2814 and extends to z-position 2816. In FIG.28B, the overlap region begins at z-position 2818 and extends toz-position 2820. When, as in FIG. 28A, the overlap region is less thansome threshold value, in one embodiment four microns, or the signalintensities within the plateau intervals differ from one another bygreater than some threshold amount, in one embodiment 2 percent, then,as shown in FIG. 28A, a left boundary Z_(l) 2822 and a right boundaryZ_(r) 2824 are chosen as the left-most and right-most boundaries of theplateau intervals. By contrast, when the width of the overlap regionexceeds a threshold value and the signal intensities within the plateauintervals differ by less than a threshold amount, then the left boundaryZ_(l) is chosen as the left-most point of the overlap 2818 and the rightboundary Z_(r) is chosen as the right-most point of the overlap region2820, as shown in FIG. 28B.

[0069] Window_focus then centers smaller windows, in one embodiment,2-micorn windows, at each z-position within the left and rightboundaries, and searches, starting from the center, to find a smallwindow with acceptable criteria most close to the center of theZ_(l)-to-Z_(r) interval. The criteria used in one embodiment, fortwo-micron narrow windows, is that the signal intensities within thewindow must differ by less than 1 percent. In one embodiment, the searchfirst considers narrow window 2902, next considers window 2904, nextconsiders window 2906, next considers window 2908, and so forth, in aping-pong like fashion. The first window considered that meets thecriteria is selected as the focus window, and the z-position of thewindow's center is selected as the focus Z_(f).

[0070]FIGS. 30 and 31 show flow-control diagrams for the routine“window_focus” and the routine “micro_focus,” called from the routine“window_focus.” Beginning with FIG. 30 in step 3002, window_focusreceives the I/z data for scans of the reference array in each channel.In step 3004, window_focus determines the z-positions for the signalintensity peaks for each channel, as discussed above with reference toFIG. 23, and checks to see that they are within the central 80-percentinterval of the full z-position range Z_(max) ⁻ to Z_(max) ⁻. If all thepeak positions are not within the 80 percent central range, asdetermined by window_focus in step 3006, then an error is returned.Otherwise, in the nested for-loops comprising steps 3008-3014,window_focus determines the plateau intervals for each channel, asdescribed above with reference to FIGS. 26-28B. Then, as discussed withreference to FIG. 29, window_focus calls the routine “micro_focus,” instep 3024, to compute the z-position corresponding to the optimal focusdistance Z_(f), returning the optimal focus z-position Z_(f) in step3026, along with a status value indicating whether or not the plateaudetermination carried out in steps 3008-3014 successfully completed.Step 3008 is the first step of the outer for-loop of the nestedfor-loops of steps 3008-3014. The outer for-loop is repeated over aseries of decreasing window sizes, as described with reference to FIGS.24-25, in order to select plateau intervals for each channel scanned bythe molecular array scanner.

[0071] The inner for-loop comprising steps 3009-3011 determines plateauintervals for each channel by the method described above with referenceto FIGS. 26 and 17. A flow-control diagram for the routine“determine_plateau_interval,” called from step 3010, is not provided, asthe technique is thoroughly described, above, with reference to FIGS. 26and 27. As noted there, plateau intervals span z-positions correspondingto the top of a mesa, and are identified as windows with essentiallyflat or nearly flat slopes that follow steeply sloped windows thatcontain the walls of the mesa.

[0072] When the plateau intervals for all channels have been determinedfor a current window size, window_focus determines, in step 3011,whether the signal variation, or differences, within each plateauinterval is less than 2%. If not, then, in step 3012, window_focusdetermines whether or not the current window size is the smallest windowsize allowed for the outer for-loop. If not, then window_focusdecrements the window size, in step 3013, and control returns to step2009, where the inner for-loop is again executed. If the smallest windowsize has been tried, then window_focus returns, as the plateau windowfor each channel, the smallest windows with the least variation insignal intensity, using an RMS-like measure to be discussed below. Oncethe plateau intervals for each channel are found, then, in step 3016,window_focus computes the overlap between plateau intervals. If theoverlap is acceptable, as determined in step 3018, then, as describedwith reference to FIG. 28B, above, window_focus sets the left and rightboundaries Z_(l) and Z_(r) respectively, to the left and rightboundaries of the overlap region, in step 3020. Otherwise, as describedabove with reference to FIG. 28A, window_focus sets the Z_(l) and Z_(r)boundary z-position values to the left-most z-position and right-mostz-position of the various plateau intervals, in step 3022. Note thatthis step may also set an error return value to indicate an errorcondition corresponding to not finding a suitable plateau window for atleast one channel. Following setting of the Z_(l) and Z_(r) boundaryz-positions in step 3020 or 3022, window_focus calls the routine“micro_focus” instep 3018.

[0073]FIG. 31 shows a flow-control diagram for the routine“micro_focus,” called from step 3018 of FIG. 30. In step 3102,micro_focus computes the center of the Z_(l)-to-Z_(r) interval Z_(c).Next, in step 3104, micro_focus applies the small windows to eachincrement within the Z_(l)-to-Z_(r) interval, starting with the centerof the Z_(l)-to-Z_(r) interval Z_(c). In one embodiment of the presentinvention, 2-micron windows are used. In step 3106, micro_focusdetermines whether there are an odd number of windows in theZ_(l)-to-Z_(r) z-position range. If so, then in step 3108, micro_focusdetermines whether the center window has acceptable characteristics. Asdiscussed above with reference to FIG. 29, acceptable characteristicsmay include a variation of signal intensities within the center windowless than 1 percent. Alternatively, a slope-based method or anothermethod for determining the degree of signal-intensity constancy withinthe window may be employed. If the center window is acceptable, then, instep 3110, micro_focus sets the z-position corresponding to the optimalfocus distance, Z_(f), to the z-position of the center of the centerwindow and returns success. Otherwise, in the for-loop comprising steps3112-3116, micro_focus carries out a ping-pong search of windows, outfrom the central z-position Z_(c), to find an acceptable window closestto the center Z_(c). The index i is set to the first window to the rightof the center, and the index j is set to index the first window to theleft of the center. In each iteration of the for-loop, the index i isincremented and the index j is decremented by 1 increment, in step 3116.In step 3113, micro_focus determines whether the window index by index iis acceptable, according to the criteria described above foracceptability of the center window in step 3108. If so, then controlflows to step 3110, where the z-position corresponding to the optimalfocus distance Z_(f) is set to the center of the window indexed byvariable i. Otherwise, if the window indexed by index j is acceptableaccording to the criteria applied to the central window in step 3108,then the z-position corresponding to the optimal focus distance Z_(f) isset to the center of the window indexed by j, in step 3110. Otherwise,if there are more windows to consider in the range Z_(l)-to-Z_(r), asdetermined in step 3115, then the indices are incremented anddecremented in step 3116 and another iteration of the for-loop iscarried out. If an acceptable window is not found following terminationof the for-loop then, in step 3118, micro_focus computes aroot-mean-square-like (“RMS-like”) value, {square root}{square root over(((max(I_(r))−min(I_(r)))/I_(rmax))²+((max(I_(g))−min(I_(g)))/I_(gmax))²)},for each window, where max(I_(r)) is maximum red-channel intensity forthe window, min(I_(r)) is the minimum red-channel intensity for thewindow, I_(rmax) is the maximum red intensity of the red-channel I/zfunction, max(I_(g)) is maximum green-channel intensity for the window,min(I_(g)) is the minimum green-channel intensity for the window, andI_(gmax) is the maximum green intensity of the green-channel I/zfunction. Micro_focus then selects the window with the smallest RMS-likevalue in step 3120. The z-position of the center of the selected windowis returned as the z-position corresponding to the optimal focusdistance Z_(f).

[0074] Although the present invention has been described in terms of aparticular embodiment, it is not intended that the invention be limitedto this embodiment. Modifications within the spirit of the inventionwill be apparent to those skilled in the art. For example, manydifferent scan patterns may be used to construct the I/z data employedby the automated focus-distance determination routines. Different rangesof window sizes may be employed, and different orderings in windowsearching may be employed. The automated focus-distance determinationroutines can be implemented in many different languages, or hardwarecircuits, in an almost limitless number of ways, using different modularorganizations and control logic. The methods was generally described inflow-control diagrams to include an arbitrary number of channels, and isintended to be applicable for 1-channel, 2-channel, and many-channelmolecular array scanners.

[0075] The foregoing description, for purposes of explanation, usedspecific nomenclature to provide a thorough understanding of theinvention. However, it will be apparent to one skilled in the art thatthe specific details are not required in order to practice theinvention. The foregoing descriptions of specific embodiments of thepresent invention are presented for purpose of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed. Obviously many modificationsand variations are possible in view of the above teachings. Theembodiments are shown and described in order to best explain theprinciples of the invention and its practical applications, to therebyenable others skilled in the art to best utilize the invention andvarious embodiments with various modifications as are suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the following claims and their equivalents:

1. A method for determining an optimal focus distance of a molecularscanner, the method comprising: scanning portions of a reference arrayover a range of positions, each position representing a differentdistance between an illuminated region of the reference array and anemitted-light detection component of the molecular array, each portionscanned at a different position; assembling data collected from scanningthe reference array into a representation of an intensity/positionfunction; and employing a focus-distance determination method todetermine an optimal focus distance or range of focus distances.
 2. Themethod of claim 1 wherein scanning portions of a reference array over arange of positions relative to an emitted-light detection componentfurther includes: for each position, scanning a set of rows, filteringthe intensity values scanned for each row, and averaging the intensityvalues for the set of rows into an average intensity value for theposition.
 3. The method of claim 1 wherein assembling data collectedfrom scanning the reference array into a representation of anintensity/position function further includes associating each positionwith an average intensity value.
 4. The method of claim 1 whereinemploying a focus-distance determination method to determine an optimalfocus distance or range of focus distances further includes employing apeak-intensity-focus-distance determination method.
 5. The method ofclaim 4 wherein the peak-intensity-focus-distance determination methodcomprises: using an intensity/position function for each channel of themolecular array scanner, determining the position of the peak intensityin the intensity/position function, and starting from the peak-intensityposition, moving right and left in position in order to identify a leftplateau position and a right plateau position at which the correspondingintensity falls below a threshold value, and selecting the positionsbetween the left plateau position and the right plateau position as theplateau interval for the channel; determining an overlap positioninterval corresponding to the overlap in position of the plateauintervals of each channel; and when the overlap position interval andplateau for each channel meet acceptance criteria, returning a positionwithin a plateau interval as the focus distance.
 6. The method of claim5 wherein the acceptance criteria comprise the overlap position intervalhaving a size greater than an overlap position interval threshold sizeand the plateau for each channel having a size greater than a plateauthreshold size.
 7. The method of claim 1 wherein employing afocus-distance determination method to determine an optimal focusdistance or range of focus distances further includes employing awindow-focus-distance determination method.
 8. The method of claim 7wherein the focus-distance determination method comprises: using anintensity/position function for each channel of the molecular arrayscanner, determining the position of the peak intensity in theintensity/position function for each channel, and returning an errorwhen the positions of peak intensity for each channel do not all fallwithin a central portion of the range of position; finding, for eachchannel, a plateau interval in the intensity/position function for thechannel; finding an overlap position interval that represents overlap inpositions from the plateau intervals for each channel; when the overlapposition interval meets a set of acceptance criteria, finding afocus-distance within the overlap position interval.
 9. The method ofclaim 8 wherein finding, for each channel, a plateau interval in theintensity/position function for the channel further includes: fordecreasing window intervals sizes, searching window intervals in theposition range of the intensity/position function for the channel for awindow interval in which intensities differ by less than a thresholdvalue, when a single window interval contains intensities that differ byless than the threshold value, returning the single window interval,when more than one window interval contains intensities that differ byless than a threshold value, selecting a window interval having theleast difference in intensities and returning the selected windowinterval, and when the current window interval size is less than aminimum window interval size, returning a default window interval. 10.The method of claim 9 wherein finding a focus-distance within theoverlap position interval further includes: starting from a centerposition within the overlap position interval, searching outward fromthe center position to find a small window interval closest to thecenter position with intensity differences less than asmall-window-final-intensity-difference threshold.
 11. Signal intensitydata scanned from the surface of a molecular array at a focus distancedetermined by the method of claim 1 encoded by: storing representationsof the signal intensity data in a machine readable medium; transmittingrepresentations of the signal intensity data over an electroniccommunications medium; displaying the signal intensity data on displaydevice; and printing representations of the signal intensity data in ahuman readable medium.
 12. A set of computer instructions for carryingout the method of claim 1 encoded by one of: storing the computerinstructions in a machine readable medium; transmitting the computerinstructions over an electronic communications medium; and printing thecomputer instructions in a human readable medium.
 13. A molecular arrayscanner comprising: a probe-molecule excitation system; an emitted-lightphotodetection system that produces a signal representative of theemitted-light intensity; a molecular-array-holding stage that holds amolecular array for scanning and that can be moved through a range ofpositions that place an illuminated region of the surface of themolecular array at different distances from the emitted-lightphotodetection system; and an automated focus-distance-determinationsubsystem.
 14. The molecular array scanner of claim 13 wherein theautomated focus-distance-determination subsystem: scans portions of areference array over a range of positions, each position representing adifferent distance between an illuminated region of the reference arrayand the emitted-light photodetection system, each portion scanned at adifferent position; assembles data collected from scanning the referencearray into a representation of an intensity/position function; andemploys a focus-distance determination method to determine an optimalfocus distance or range of focus distances.
 15. The molecular arrayscanner of claim 14 wherein the automated focus-distance-determinationsubsystem scans portions of a reference array over a range of positionsby: for each position, scanning a set of rows, filtering the intensityvalues scanned for each row, and averaging the intensity values for theset of rows into an average intensity value for the position.
 16. Themolecular array scanner of claim 15 wherein the automatedfocus-distance-determination subsystem assembles data collected fromscanning the reference array into a representation of anintensity/position function by associating each position with an averageintensity value.
 17. The molecular array scanner of claim 15 wherein theautomated focus-distance-determination subsystem employs afocus-distance determination method to determine an optimal focusdistance or range of focus distances by employing apeak-intensity-focus-distance determination method.
 18. The moleculararray scanner of claim 15 wherein the automatedfocus-distance-determination subsystem employs a focus-distancedetermination method to determine an optimal focus distance or range offocus distances by employing a window-focus-distance determinationmethod.
 19. Signal intensity data scanned from the surface of amolecular array at a focus distance determined by the molecular arrayscanner of claim 13 encoded by: storing representations of the signalintensity data in a machine readable medium; transmitting representationof the signal intensity data over an electronic communications medium;displaying the signal intensity data on display device; and printingrepresentations of the signal intensity data in a human readable medium.20. A set of computer instructions that implement themolecular-array-scanner automated focus-distance-determination subsystemof claim 1 encoded by one of: storing the computer instructions in amachine readable medium; transmitting the computer instructions over anelectronic communications medium; and printing the computer instructionsin a human readable medium.